Biologically active compounds conjugated with polyoxyalkylenes (such as polyethylene glycol) can provide enhanced biocompatibility for the compound. See, for example, U.S. Pat. Nos. 5,366,735 and 6,280,745. A review of this subject by Zalipsky, in Bioconjugate Chem., 1995, 6, p150-165, identified polyethylene glycol as one of the best biocompatible polymers to conjugate with a biologically active compound (such as a drug, a protein, a peptide or an enzyme) to produce a conjugate having improved properties such as compatible solubility characteristics, reduced toxicity, improved surface compatibility, increased circulation time and reduced immunogenicity.
Polyethylene glycol (PEG) is a linear polyoxyalkylene terminated at the ends thereof with hydroxyl groups and generally represented by the formula: HO(CH2CH2O)nH. As discussed by Henmanson in Chapter 15 of Bioconjugate Techniques (1996), monomethyl polyethylene glycol (mPEG) generally represented by the formula: CH3O(CH2CH2O)nH, is often used to prepare a polyethylene glycol conjugate with a biologically active compound typically by way of a coupling reaction between the biologically active compound and a derivative of MPEG such as mPEG p-nitrophenyl carbonate. A biologically active compound coupled to PEG is said in the art to be “PEGylated”.
As discussed by Seely and Richey, J. of Chrom. A, 908 (2001) 235-241 herein fully incorporated by reference, the serum half-life of a PEGylated biopharmaceutical agent increases as the molecular weight of the PEG chain is increased. One means of increasing the molecular weight of a PEG derivatizing agent is to form a “multi-armed” PEG derivative. A multi-armed PEG derivative comprises a plurality of PEG chains thereby increasing the molecular weight of the multi-armed PEG derivative. A multi-armed PEG derivative can be multi-functional (as discussed by Seely and Richey, by WO 97/32607 and by USPAP 2003/0065134 each of which are herein fully incorporated by reference) or mono-functional (as discussed by U.S. Pat. Nos. 5,919,455 and 5,932,462 each of which are herein fully incorporated by reference).
The prior art methods for making mPEG carbonates, such as mPEG p-nitrophenyl carbonate, are set forth in U.S. Pat. No. 5,286,637, by Fortier et al. Applied Biochemistry (1993) 17(1), 115-130 and by Veronese et al., Applied Biochem. Biotech. 11, 141-152 (1985). U.S. Pat. No. 5,286,637 is based on the reaction of mPEG with p-nitrophenyl chloroformate in a solvent of methylene chloride containing triethylamine. Fortier et al. used pyridine instead of triethylamine. Veronese et al. used acetonitrile instead of methylene chloride. All of these prior art methods for making MPEG p-nitrophenyl carbonate also produce an impurity (believed to be the amine salt of the hydrolyzed MPEG p-nitrophenyl carbonate) when the concentration of the MPEG p-nitrophenyl carbonate is relatively high, which impurity is difficult to separate from the mPEG p-nitrophenyl carbonate. Thus, there remains a need for an improved method for making mPEG carbonates (such as MPEG p-nitrophenyl carbonate) at relatively high concentration, which method never-the-less produces less impurity.